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Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa

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Abstract

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC 6.2.1.12), which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation.

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Abbreviations

CCR:

Cinnamoyl-CoA reductase

4CL:

4-Coumarate-CoA ligase

COMT:

Caffeate O-methyltransferase

CAD:

Cinnamyl alcohol dehydrogenase

HCA:

Hydroxycinnamic acid

HPLC-MS:

High performance liquid chromatography-mass spectrometry

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Acknowledgments

The work was jointly supported by the National High Technology Research and Development (863 Program 2011AA100203), National Natural Science Foundation Project (Grant No. 30630053) to Dr. Xiangning Jiang and Project of Beijing Forestry University Young Scientist Fund (No.2010BLX03) to Dr. Ying Gai.

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Authors and Affiliations

  1. College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, People’s Republic of China

    Botao Han, Shaoli Liu, Ying Gai & Xiangning Jiang

  2. The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration, National Engineering Laboratory for Tree Breeding, Beijing, 100083, People’s Republic of China

    Xiangning Jiang

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  1. Botao Han
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  2. Shaoli Liu
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Correspondence to Xiangning Jiang.

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Han, B., Liu, S., Gai, Y. et al. Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa . J. Plant Biochem. Biotechnol. 23, 348–357 (2014). https://doi.org/10.1007/s13562-013-0218-6

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